This project will investigate the role of human antibodies in protection against or enhancement of infection caused by serotype 3 dengue viruses. Pre-existing heterotypic dengue antibody is a risk factor for lifethreatening severe illness, designated dengue hemorrhagic fever (DHF). Although it is well established that the induction of heterotypic dengue antibodies can predispose subjects to DHF, the molecular basis for this problem is not well understood. This issue is a major obstacle to effective development of dengue vaccines as it is not clear that tetravalent vaccine formulations can be established that always retain the immunogenicity of all four serotypes. Better knowledge of the molecular basis of antibody-mediated neutralization of infection is needed. In the work proposed in this application, we will derive large panels of human monoclonal antibodies directed to dengue virus serotype 3 from the B cells of subjects previously infected with that virus. In previous work funded by the RCE, we have developed a very reliable and robust method for generating human monoclonal antibody secretign hybridoma lines from immune donors. We will determine the genetic and structural basis for effective neutralization of dengue viruses by sequence analysis of antibodies, generation of recombinant antibodies for study of naturally occurring somatic mutations, generation of escape mutant viruses, definition of determinants of components of affinity (on and off rates), and determination of structures of immunodominant antibodies bound to viral antigens.